The subject invention relates to enzymatic processes for the preparation of specialty chemicals, e.g., for the preparation of fructose (levulose). Those skilled in this art have found that immobilization of a particular enzyme which catalyzes an enzymatic reaction, whether in the whole microbial cell or in cell-free systems, e.g. the enzyme or cellular material containing the enzyme, generally results in better yield of the desired product and an improvement in enzyme stability. This immobilization step can be accomplished by a number of procedures well known to those skilled in the art. Patents relating to the immobilization of enzymes can be exemplified as follows:
U.S. Pat. No. 3,779,869--Glucose isomerase within bacterial cells can be stabilized by glutaraldehyde treatment. PA0 U.S. Pat. No. 3,935,068--Cationic polyelectrolytes are used for enzyme immobilization. PA0 U.S. Pat. No. 3,980,521--Microbial cells are concentrated and cross-linked with glutaraldehyde to form a coherent solid product. PA0 U.S. Pat. No. 4,288,552--Intracellular glutaraldehyde sensitive enzymes are immobilized by reacting microbial cell material with glutaraldehyde in the presence of a polyamine.
An important part of the immobilization process frequently involves the flocculation of microbial cells or cellular material, typically followed by cross-linking of the resulting flocculated cells or cellular material in order to obtain an immobilized enzyme which is easier to work with and which has improved enzyme stability (see U.S. Pat. Nos. 4,337,313; 3,821,086; 3,989,596; 3,935,068 and 3,935,069).
Flocculation is used to increase the aggregation of small particulate matter, such as microbial cells, cellular materials containing enzymes or the enzymes themselves, contained in an aqueous environment, for example, through coacervation or precipitation. Increasing the aggregation of the small particulate matter facilitates removal of the water. Although a flocculation step is generally unnecessary, the greater ease of water removal prior to cross-linking which can be accomplished by employing a flocculation step typically makes a flocculation step prior to cross-linking desirable.
Flocculation of microbial cells or cellular materials can be accomplished by use of a cationic polyelectrolyte, for example, polyamines, cationic polyaminoacids, cationic polyacrylamides, cationic poly(vinyl chloride), cationic copolymers, and cationic flocculants.
Cross-linking is used to impart more dimensional stability to the microbial cells or cellular materials being cross-linked. The prior art discloses the use of various di- or multi-functional cross-linking reagents. Few of these reagents are useful in large scale applications. Currently, glutaraldehyde is the cross-linker of choice due to its low cost, high reactivity and the good stability of its end products. (See U.S. Pat. Nos. 3,980,521, 4,288,552 and 4,355,105). However, glutaraldehyde has some drawbacks. Generally, the loss of enzyme activity resulting from glutaraldehyde cross-linking is significant even under mild conditions. Thus, there exists a need for milder and more specific cross-linkers for enzyme applications. Further, if the flocculation and cross-linking steps can be performed by a single reagent, obvious economic advantages in the immobilization process would be realized.